Identification and characterization of probiotic yeast isolated from digestive tract of ducks

Identification and characterization of probiotic yeast isolated from digestive tract of ducks ABSTRACT The objective of this study was to isolate and identify yeast strains from the digestive tract of ducks, and evaluate in vitro their potential as probiotics in poultry. The yeast strains were isolated using malt extract agar medium, and identified through morphological, physiological, and biochemical tests as well as sequence homology analyses of 26S rDNA D1/D2 region. A total of 35 yeast strains were isolated from the guts of Cherry Valley meat ducks, including seven strains of Saccharomyces cerevisiae (S. cerevisiae). These seven strains of S. cerevisiae were further screened for their use as alternative yeast probiotics strains for poultry feed. The yeast strains were characterized for their cell surface hydrophobicity, autoaggregation ability, and resistance to high temperature (30°C, 37°C, and 42°C), low pH (2.0, 3.0, and 4.0), bile salts (0.3% and 0.6%), and nutrition starvation (2, 4, 6, 8, 10, and 12 days). The isolates of WHY-2 and WHY-7 had a higher survival percentage at 37°C, pH 2.0, 0.60% poultry bile salts, and 10 days of nutrition starvation, with higher cell surface hydrophobicity and autoaggregation, when compared with the other isolates, suggesting that the isolates WHY-2 and WHY-7, could be used as probiotic candidates. The data obtained in this study could help in selecting probiotic yeast candidates for use in poultry industry. INTRODUCTION Growth-promoting antimicrobials, such as ionophore antibiotics, have been widely utilized and are still being used in some countries. However, owing to the increasing safety concerns about the risk of development of antibiotics resistance and persistence of chemical residues in animal products, some strategies based on supplementation of more “natural” products such as probiotics, have been established to improve herd health and productivity (Francisco et al., 2009). Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2006). Several criteria are required for a microorganism to be considered as a probiotic: it must be innocuous, causing neither toxicity nor illness to the host, it must occur in high amounts in the product with which it is administered and maintain its viability during the product's shelf-life, and it must be technologically usable and survive gastrointestinal (GI) transit (Hill et al., 2014; Larsen et al., 2014). Yeasts are one of the major groups of probiotics (Czerucka and Rampal, 2002). In the past decades, yeasts have been considered as one of the microorganisms that could be used in probiotics for poultry (Fleet, 2007; Jacques and Casaregola, 2008). Yeasts have unique properties, such as not being affected by antibacterial treatments, which can help to alleviate antibiotic-associated diarrhea (Moslehijenabian et al., 2010; Hatoum et al., 2012). Currently, the conventional yeast Saccharomyces cerevisiae is the most common animal probiotic yeast available on the market (Czerucka et al., 2007; Didari et al., 2014). However, the effects of different yeast strains on animals are significantly different. It is generally believed that strains isolated from the animal digestive ecosystem are adapted to the GI tract environment and could be used as ideal probiotics (Hume et al., 2012). Nevertheless, each strain must be well identified and characterized in vitro and in vivo (FAO/WHO, 2006) before being used for these purposes. The in vitro selection criteria for probiotic candidates include tolerance to GI tract conditions and ability of the candidate strains to adhere to intestinal epithelial cells (Trotman, 2002; Van et al., 2005). To reach the target site of action alive, the potential probiotic microorganisms must be able to tolerate a variety of adverse conditions of the GI tract, such as temperature stress (i.e., internal body temperature of 37°C), acidic gastric juice, bile salts, and nutrition starvation (Kumura et al., 2004; Van et al., 2005). Hence, selection of probiotic microorganisms is typically performed in vitro by screening the probiotic candidates for survival under simulated GI tract conditions (Martins et al., 2008). Cell-surface hydrophobicity and autoaggregation are important physicochemical forces that help in the adherence of cells to biological and abiotic surfaces, and may be the main factors determining the adhesion properties of cells, which may also be the basis for probiotics to function as a biological barrier (Van et al., 2005). Thus, the objective of the present study was to isolate, identify, and screen (in vitro) yeast strains (S. cerevisiae) from duck GI tracts for their probiotic potential in the poultry industry. MATERIALS AND METHODS Yeast Isolation Procedures A total of 24 healthy ducks were obtained from duck farm and farmers’ market in Yichang and Wuhan, respectively, and yeast strains were isolated from these ducks’ GI tracts. In brief, the ducks’ digestive tract contents were extracted. Each sample was diluted 10-fold in sterile saline (0.85% NaCl) and incubated at ambient temperature for 90 min. Serial dilutions of each sample were performed and 1 mL aliquots of appropriate dilutions were inoculated (in duplicates) by the pour-plate method on yeast peptone dextrose (YPD) agar plates (containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 20 g/L agar) supplemented with chloramphenicol (200 mg/L) and incubated at 30°C for 48 h (Kurtzman, 2011). Subsequently, colonies showing different morphologies were selected and inoculated thrice on malt extract agar plates. The pure cultures were preserved on Sabouraud agar plates (Scharlau, Spain) at 8 ± 2°C until further use. Characterization of Yeast Strains All the yeasts were preliminarily grouped based on their cultural morphology, urease production, and physiological characteristics such as carbon assimilation, nitrogen sources, and amyloid compounds production (Yarrow, 1998). The yeast strains were cultured in YPD broth for 24 h at 30°C. The total genomic DNA of the yeast strains were extracted and purified by using a yeast genomic DNA extraction kit (Tiangen Biotech Co. Ltd, Beijing, China). The D1/D2 region of the large subunit of 26S rDNA of the selected yeast strains was amplified with universal primers, 5′-GCATATCAATAAGCGGAGGAAAAG-3′ (forward) and 5′-GGTCCGTGTTTCAAGACGG-3′ (reverse) (Kurtzman and Robnett, 1998). The primers were synthesized by Sangon Biotech Co. Ltd, Shanghai, China. PCR was performed in a 50 μL reaction system consisting of 25 μL of 2 × Taq PCR Master Mix (Tiangen Biotech Co. Ltd), 4 μL of DNA template, 1 μL of each primer, and 19 μL of sterile deionized H2O. The reaction conditions were as follows: initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 45 s, renaturation at 55°C for 1 min, and extension at 72°C for 70 s, and final extension at 72°C for 10 min. The purity of the PCR products was observed with 1% agarose gel electrophoresis. The sequencing of the 26S rDNA gene was performed by Sangon Biotech Co. Ltd. The yeast strains were identified after aligning the sequences to a nonredundant database (www.ncbi.nlm.nih.gov/blast). The strains of S. cerevisiae were identified for further investigation as candidate probiotics. Tolerance to High Temperature Sterile YPD broths were inoculated with 1% (v/v) 18 h culture broth of the test isolate and incubated at 30°C, 37°C, or 42°C. After 30 min, samples were collected and the viable cells counts (CFU/mL) were determined (Garcıa-Hernandez et al., 2012). The assay was performed in triplicate and the survival percentage (S) was calculated as follows:   \[ {\rm{S}}(\%) = \left( {\rm{N}}_{\rm{t}} \times 100 \right)/{{\rm{N}}_0} \] where Nt is CFU/mL at temperature t = 37°C or 42°C, and N0 is CFU/mL at 30°C. Tolerance to Low pH A suspension of the test isolate was prepared in YPD broth and its optical density at 600 nm (OD600) was adjusted to 1.0. Then, the suspension was centrifuged at 5000 × g for 10 min, washed with buffered phosphate solution (10 mM, pH 7.4), and resuspended in 3 mL of buffered solution phosphate solution with pH adjusted to 2.0, 3.0, or 4.0 with 90% lactic acid. After 24 h of incubation, the samples were serially diluted and inoculated onto YPD agar plates to determine the cells counts. The experiment was performed in triplicate according to completely randomized design, and the survival percentage (S) was calculated as follows:   \[ {\rm{S}}(\%) = {\rm{[(CFU/mL)}}_{{\rm{YPD }} + {\rm{Lactic\,\,acid}}} \times 100]/{{\rm{(CFU/mL)}}_{{\rm{YPD}}}}\] Tolerance to Bile Salts A suspension of the test isolate was prepared in YPD broth and its OD600 was adjusted to 1.0. Then, the suspension was serially diluted, inoculated onto Sabouraud agar plates containing 0%, 0.30%, or 0.60% (w/v) poultry bile salts (BoMei Biotech Co. Ltd, Shanghai, China), incubated at 30°C for 72 h, and the cell counts (CFU/mL) for each treatment were determined. The assay was performed in triplicate and the survival percentage (S) was calculated as follows:   \[ {\rm{S}}(\%) = [\rm{(CFU/mL)}_{\rm YPD+Salts} \times 100] /{{\rm (CFU/mL)}_{\rm YPD} } \] Tolerance to Nutrition Starvation A suspension of the test isolate was prepared in YPD broth and its OD600 was adjusted to 1.0. Then, 1% (v/v) suspension was inoculated into sterile water. After 2, 4, 6, 8, 10, or 12 days of incubation at 30°C, the suspensions were re-inoculated into YPD broth and incubated at 30°C for 72 h for growth experiment. The cell counts (CFU/mL) for each treatment were determined. Hydrophobicity of the Cell Surface The surface hydrophobicity of the S. cerevisiae strains was determined based on the adhesive capability of the yeast strains to hydrocarbon compounds (Holle et al., 2012). The yeast cells (5 × 106 cells/mL) were suspended in phosphate buffer (pH 7.1) containing 0.2 g/L MgSO4 and 1.8 g/L urea. Then, the cell suspension (10.0 mL) was placed in standard glass test tubes and acid-washed. Subsequently, n-hexadecane (2 mL) was added to each tube, vortex-mixed for 2 min, and left to stand for 15 min to ensure complete separation of the two phases. The aqueous phase was carefully removed and its absorbance was determined at 600 nm. The hydrophobicity of the cell surface (%) was calculated according to the following equation:   \[ {\rm{Hydrophobicity}}(\%) = \left( {{\rm{1}} - {\rm{A/}}{{\rm{A}}_{\rm{0}}}} \right) \times {\rm{100}} \] where A0 is the initial absorbance of the cell suspension, and A is the absorbance of the aqueous phase after mixing. Autoaggregation To measure the autoaggregation percentage, 80 mL of the YPD culture broth of the test isolate were harvested by centrifugation (4000 × g, 5 min) and washed twice with a sterile solution of 0.9% NaCl. The resulting pellet was resuspended in 5 mL of 0.9% NaCl solution and transferred to a disposable plastic cuvette. Subsequently, the OD600 of the suspension was measured at 0, 2, 4, and 24 h. The autoaggregation percentage was calculated as follows:   \[ {\rm{Autoaggregation }}(\%) = \left( {{\rm{1}} - {\rm{A/}}{{\rm{A}}_{\rm{0}}}} \right) \times {\rm{100}} \] where At represents the OD at time t = 2, 4, or 24 h, and A0 represents the OD at t = 0 h. Statistical Analysis The data obtained were subjected to analysis of variance and the significant differences were compared using Duncan's multiple range test. Values of P < 0.05 indicated statistically significant differences. The data analyses were performed with SPSS software (ver 22.0; IBM, Armonk, NY, USA). All the results are presented as the mean with standard deviation of triplicate values. RESULTS Isolation and Identification of Yeast Strains A total of 35 yeast colonies with different macroscopic morphologies were isolated from duck GI tracts. After comparing the sequences of D1/D2 region of 26S rDNA extracted from the yeast strains with those in the GenBank database, the strains were identified as Candida albicans (Y-1, Y-2, Y-3, Y-6, Y-9, Y-10, Y-15, Y-16, Y-21, Y-30), Cryptococcus arboriformis (Y-4, Y-22), Kazachstania bovina (Y-5, Y-7, Y-17, Y-23), Candida parapsilosis (Y-8, Y-11, Y-14, Y-24, Y-26, Y-27, Y-28), Lodderomyces elongisporus (Y-12, Y-35), Kodamaea ohmeri (Y-13), S. cerevisiae (Y-18, Y-19, Y-20, Y-25, Y-31, Y-33, Y-34), and Candida glabrata (Y-29, Y-32) (Table 1). As S. cerevisiae is the most common animal probiotic yeast, the seven strains of S. cerevisiae were chosen for further investigation and renamed as WHY-1, WHY-2, WHY-3, WHY-4, WHY-5, WHY-6, and WHY-7. The sequences of the D1/D2 region of 26S rDNA of these seven strains have been submitted to GenBank (Table 2). The biochemical identification results showed that all the S. cerevisiae strains were able to ferment D-glucose, maltose, and sucrose, but not lactose. Furthermore, all the strains, except WHY-4, could ferment galactose. With regard to melibiose fermentation, except WHY-2 and WHY-6, the rest of the strains showed positive results. Moreover, while none of the strains could assimilate potassium nitrate, L-lysine, and cadaverine, they could grow in non-niacin YPD agar. In addition, WHY-2, WHY-3, and WHY-6 were positive in the non-pyridoxine test, whereas WHY-2, WHY-4, and WHY-5 were positive in the non-thiamine test. With regard to 0.01% cycloheximide tests, starch-like compound formation test, urea decomposition test, and 50% D-glucose growth test, the results were negative for all the seven strains (Table 3). Table 1. Molecular identification of the yeast strains isolated from duck gastrointestinal tracts by sequencing of the D1/D2 region of 26S rDNA gene. Strains  Molecular identification  Similarity (%)  GenBank ID of type strain  Y-1, 2, 3, 6, 9, 10, 15, 16, 21, 30  Candida albicans  99.0  KJ534502  Y-4, 22  Cryptococcus arboriformis  99.0  AB260936  Y-5, 7, 17, 23  Kazachstania bovina  99.5  AB499993  Y-8, 11, 14, 24, 26, 27, 28  Candida parapsilosis  99.0  FJ746059  Y-12, 35  Lodderomyces elongisporus  100.0  LC195009  Y-13  Kodamaea ohmeri  99.0  GU597323  Y-18, 19, 20, 25, 31, 33, 34  Saccharomyces cerevisiae  99.6  KM655848  Y-29, 32  Candida glabrata  99.5  HM627130  Strains  Molecular identification  Similarity (%)  GenBank ID of type strain  Y-1, 2, 3, 6, 9, 10, 15, 16, 21, 30  Candida albicans  99.0  KJ534502  Y-4, 22  Cryptococcus arboriformis  99.0  AB260936  Y-5, 7, 17, 23  Kazachstania bovina  99.5  AB499993  Y-8, 11, 14, 24, 26, 27, 28  Candida parapsilosis  99.0  FJ746059  Y-12, 35  Lodderomyces elongisporus  100.0  LC195009  Y-13  Kodamaea ohmeri  99.0  GU597323  Y-18, 19, 20, 25, 31, 33, 34  Saccharomyces cerevisiae  99.6  KM655848  Y-29, 32  Candida glabrata  99.5  HM627130  View Large Table 1. Molecular identification of the yeast strains isolated from duck gastrointestinal tracts by sequencing of the D1/D2 region of 26S rDNA gene. Strains  Molecular identification  Similarity (%)  GenBank ID of type strain  Y-1, 2, 3, 6, 9, 10, 15, 16, 21, 30  Candida albicans  99.0  KJ534502  Y-4, 22  Cryptococcus arboriformis  99.0  AB260936  Y-5, 7, 17, 23  Kazachstania bovina  99.5  AB499993  Y-8, 11, 14, 24, 26, 27, 28  Candida parapsilosis  99.0  FJ746059  Y-12, 35  Lodderomyces elongisporus  100.0  LC195009  Y-13  Kodamaea ohmeri  99.0  GU597323  Y-18, 19, 20, 25, 31, 33, 34  Saccharomyces cerevisiae  99.6  KM655848  Y-29, 32  Candida glabrata  99.5  HM627130  Strains  Molecular identification  Similarity (%)  GenBank ID of type strain  Y-1, 2, 3, 6, 9, 10, 15, 16, 21, 30  Candida albicans  99.0  KJ534502  Y-4, 22  Cryptococcus arboriformis  99.0  AB260936  Y-5, 7, 17, 23  Kazachstania bovina  99.5  AB499993  Y-8, 11, 14, 24, 26, 27, 28  Candida parapsilosis  99.0  FJ746059  Y-12, 35  Lodderomyces elongisporus  100.0  LC195009  Y-13  Kodamaea ohmeri  99.0  GU597323  Y-18, 19, 20, 25, 31, 33, 34  Saccharomyces cerevisiae  99.6  KM655848  Y-29, 32  Candida glabrata  99.5  HM627130  View Large Table 2. GenBank accession numbers for the D1/D2 region of 26S rDNA of the S. cerevisiae strains from duck gastrointestinal tracts. Strains  Accession #  WHY -1  MG641146  WHY -2  MG641147  WHY -3  MG641148  WHY -4  MG641149  WHY -5  MG641150  WHY -6  MG641151  WHY -7  MG641152  Strains  Accession #  WHY -1  MG641146  WHY -2  MG641147  WHY -3  MG641148  WHY -4  MG641149  WHY -5  MG641150  WHY -6  MG641151  WHY -7  MG641152  View Large Table 2. GenBank accession numbers for the D1/D2 region of 26S rDNA of the S. cerevisiae strains from duck gastrointestinal tracts. Strains  Accession #  WHY -1  MG641146  WHY -2  MG641147  WHY -3  MG641148  WHY -4  MG641149  WHY -5  MG641150  WHY -6  MG641151  WHY -7  MG641152  Strains  Accession #  WHY -1  MG641146  WHY -2  MG641147  WHY -3  MG641148  WHY -4  MG641149  WHY -5  MG641150  WHY -6  MG641151  WHY -7  MG641152  View Large Table 3. Biochemical identification of the S. cerevisiae strains from duck gastrointestinal tracts.   Yeast isolates, WHY  Substrate  1  2  3  4  5  6  7  D-Glucose  +  +  +  +  +  +  +  Maltose  +  +  +  +  +  +  +  Galactose  +  +  +  −  +  +  +  Lactose  −  −  −  −  −  −  −  Melibiose  +  −  +  +  +  −  +  Saccharose  +  +  +  +  +  +  +  Niter  −  −  −  −  −  −  −  L-Lysine  −  −  −  −  −  −  −  Cadaverine  −  −  −  −  −  −  −  Non-niacin  +  +  +  +  +  +  +  Non-pyridoxine  −  +  +  −  −  +  −  Non-thiamine  −  +  −  +  +  −  −  0.01% Cycloheximide  −  −  −  −  −  −  −  starch-like compound formation  −  −  −  −  −  −  −  urea decomposition  −  −  −  −  −  −  −  50% D-glucose growth  −  −  −  −  −  −  −  Hypha or pseudohypha  −  +  −  +  −  +  +    Yeast isolates, WHY  Substrate  1  2  3  4  5  6  7  D-Glucose  +  +  +  +  +  +  +  Maltose  +  +  +  +  +  +  +  Galactose  +  +  +  −  +  +  +  Lactose  −  −  −  −  −  −  −  Melibiose  +  −  +  +  +  −  +  Saccharose  +  +  +  +  +  +  +  Niter  −  −  −  −  −  −  −  L-Lysine  −  −  −  −  −  −  −  Cadaverine  −  −  −  −  −  −  −  Non-niacin  +  +  +  +  +  +  +  Non-pyridoxine  −  +  +  −  −  +  −  Non-thiamine  −  +  −  +  +  −  −  0.01% Cycloheximide  −  −  −  −  −  −  −  starch-like compound formation  −  −  −  −  −  −  −  urea decomposition  −  −  −  −  −  −  −  50% D-glucose growth  −  −  −  −  −  −  −  Hypha or pseudohypha  −  +  −  +  −  +  +  +: positive; −: negative. View Large Table 3. Biochemical identification of the S. cerevisiae strains from duck gastrointestinal tracts.   Yeast isolates, WHY  Substrate  1  2  3  4  5  6  7  D-Glucose  +  +  +  +  +  +  +  Maltose  +  +  +  +  +  +  +  Galactose  +  +  +  −  +  +  +  Lactose  −  −  −  −  −  −  −  Melibiose  +  −  +  +  +  −  +  Saccharose  +  +  +  +  +  +  +  Niter  −  −  −  −  −  −  −  L-Lysine  −  −  −  −  −  −  −  Cadaverine  −  −  −  −  −  −  −  Non-niacin  +  +  +  +  +  +  +  Non-pyridoxine  −  +  +  −  −  +  −  Non-thiamine  −  +  −  +  +  −  −  0.01% Cycloheximide  −  −  −  −  −  −  −  starch-like compound formation  −  −  −  −  −  −  −  urea decomposition  −  −  −  −  −  −  −  50% D-glucose growth  −  −  −  −  −  −  −  Hypha or pseudohypha  −  +  −  +  −  +  +    Yeast isolates, WHY  Substrate  1  2  3  4  5  6  7  D-Glucose  +  +  +  +  +  +  +  Maltose  +  +  +  +  +  +  +  Galactose  +  +  +  −  +  +  +  Lactose  −  −  −  −  −  −  −  Melibiose  +  −  +  +  +  −  +  Saccharose  +  +  +  +  +  +  +  Niter  −  −  −  −  −  −  −  L-Lysine  −  −  −  −  −  −  −  Cadaverine  −  −  −  −  −  −  −  Non-niacin  +  +  +  +  +  +  +  Non-pyridoxine  −  +  +  −  −  +  −  Non-thiamine  −  +  −  +  +  −  −  0.01% Cycloheximide  −  −  −  −  −  −  −  starch-like compound formation  −  −  −  −  −  −  −  urea decomposition  −  −  −  −  −  −  −  50% D-glucose growth  −  −  −  −  −  −  −  Hypha or pseudohypha  −  +  −  +  −  +  +  +: positive; −: negative. View Large High Temperature Tolerance The high temperature tolerance of the yeast strains is presented in Table 4. At 37°C, the survival of strains WHY-2, WHY-6, and WHY-7 was significantly higher, when compared with that of the other four strains. Furthermore, at 42°C, the survival of WHY-2 and WHY-7 was significantly higher, when compared with that of the rest of the strains, indicating that WHY-2 and WHY-7 exhibited high temperature tolerance (at both 37°C and 42°C). Table 4. Survival percentage (%) of S. cerevisiae strains incubated at 30°C, 37°C, 42°C, and 48°C for 30 min.   Temperature    Strains  30°C1  37°C  42°C  48°C  WHY -1  100.00 ± 0.00a  67.98 ± 3.51c  57.30 ± 2.37e  3.93 ± 0.97c  WHY -2  100.00 ± 0.00a  95.37 ± 3.20a  90.74 ± 3.20a,b  9.25 ± 0.61a  WHY -3  100.00 ± 0.00a  70.62 ± 1.95c  86.44 ± 4.08b,c  5.65 ± 0.98b,c  WHY -4  100.00 ± 0.00a  80.29 ± 3.34b  75.18 ± 3.32d  7.30 ± 0.26a,b  WHY -5  100.00 ± 0.00a  72.77 ± 4.30b,c  71.96 ± 3.83d  7.59 ± 0.77a,b  WHY -6  100.00 ± 0.00a  91.60 ± 5.25a  80.67 ± 3.36c,d  5.88 ± 0.45b,c  WHY -7  100.00 ± 0.00a  98.04 ± 5.39a  93.14 ± 4.12a  7.84 ± 0.70a,b    Temperature    Strains  30°C1  37°C  42°C  48°C  WHY -1  100.00 ± 0.00a  67.98 ± 3.51c  57.30 ± 2.37e  3.93 ± 0.97c  WHY -2  100.00 ± 0.00a  95.37 ± 3.20a  90.74 ± 3.20a,b  9.25 ± 0.61a  WHY -3  100.00 ± 0.00a  70.62 ± 1.95c  86.44 ± 4.08b,c  5.65 ± 0.98b,c  WHY -4  100.00 ± 0.00a  80.29 ± 3.34b  75.18 ± 3.32d  7.30 ± 0.26a,b  WHY -5  100.00 ± 0.00a  72.77 ± 4.30b,c  71.96 ± 3.83d  7.59 ± 0.77a,b  WHY -6  100.00 ± 0.00a  91.60 ± 5.25a  80.67 ± 3.36c,d  5.88 ± 0.45b,c  WHY -7  100.00 ± 0.00a  98.04 ± 5.39a  93.14 ± 4.12a  7.84 ± 0.70a,b  Mean values ± standard deviation. a-eMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at 30°C had been used as a control, measured with 100%. View Large Table 4. Survival percentage (%) of S. cerevisiae strains incubated at 30°C, 37°C, 42°C, and 48°C for 30 min.   Temperature    Strains  30°C1  37°C  42°C  48°C  WHY -1  100.00 ± 0.00a  67.98 ± 3.51c  57.30 ± 2.37e  3.93 ± 0.97c  WHY -2  100.00 ± 0.00a  95.37 ± 3.20a  90.74 ± 3.20a,b  9.25 ± 0.61a  WHY -3  100.00 ± 0.00a  70.62 ± 1.95c  86.44 ± 4.08b,c  5.65 ± 0.98b,c  WHY -4  100.00 ± 0.00a  80.29 ± 3.34b  75.18 ± 3.32d  7.30 ± 0.26a,b  WHY -5  100.00 ± 0.00a  72.77 ± 4.30b,c  71.96 ± 3.83d  7.59 ± 0.77a,b  WHY -6  100.00 ± 0.00a  91.60 ± 5.25a  80.67 ± 3.36c,d  5.88 ± 0.45b,c  WHY -7  100.00 ± 0.00a  98.04 ± 5.39a  93.14 ± 4.12a  7.84 ± 0.70a,b    Temperature    Strains  30°C1  37°C  42°C  48°C  WHY -1  100.00 ± 0.00a  67.98 ± 3.51c  57.30 ± 2.37e  3.93 ± 0.97c  WHY -2  100.00 ± 0.00a  95.37 ± 3.20a  90.74 ± 3.20a,b  9.25 ± 0.61a  WHY -3  100.00 ± 0.00a  70.62 ± 1.95c  86.44 ± 4.08b,c  5.65 ± 0.98b,c  WHY -4  100.00 ± 0.00a  80.29 ± 3.34b  75.18 ± 3.32d  7.30 ± 0.26a,b  WHY -5  100.00 ± 0.00a  72.77 ± 4.30b,c  71.96 ± 3.83d  7.59 ± 0.77a,b  WHY -6  100.00 ± 0.00a  91.60 ± 5.25a  80.67 ± 3.36c,d  5.88 ± 0.45b,c  WHY -7  100.00 ± 0.00a  98.04 ± 5.39a  93.14 ± 4.12a  7.84 ± 0.70a,b  Mean values ± standard deviation. a-eMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at 30°C had been used as a control, measured with 100%. View Large Survival at Low pH Table 5 shows the survival of the yeast strains at pH 5.5, 4.0, 3.0, and 2.0. Strains WHY-2 and WHY-7 exhibited higher survival at low pH, when compared with the rest of the strains. The survival of WHY-2 and WHY-7 was >90% at pH 2.0, >95% at pH 3.0, and >98% at pH 4.0. Table 5. Survival percentage (%) of S. cerevisiae strains incubated at low pH for 24 h.   pH  Strains  5.51  4.0  3.0  2.0  WHY -1  100.00 ± 0.00a  93.92 ± 2.78b  84.15 ± 4.23b  72.79 ± 3.54c  WHY -2  100.00 ± 0.00a  98.75 ± 3.05a  95.57 ± 3.16a  90.29 ± 2.71a  WHY -3  100.00 ± 0.00a  88.46 ± 4.28c  78.81 ± 2.98c  67.37 ± 3.52d  WHY -4  100.00 ± 0.00a  92.39 ± 2.84b  86.14 ± 3.08b  72.20 ± 4.03c  WHY -5  100.00 ± 0.00a  90.95 ± 3.69b  85.46 ± 4.62b  78.42 ± 2.46b  WHY -6  100.00 ± 0.00a  91.85 ± 3.82b  81.02 ± 3.69b,c  64.65 ± 4.03d  WHY -7  100.00 ± 0.00a  98.90 ± 4.64a  95.90 ± 3.87a  90.30 ± 3.89a    pH  Strains  5.51  4.0  3.0  2.0  WHY -1  100.00 ± 0.00a  93.92 ± 2.78b  84.15 ± 4.23b  72.79 ± 3.54c  WHY -2  100.00 ± 0.00a  98.75 ± 3.05a  95.57 ± 3.16a  90.29 ± 2.71a  WHY -3  100.00 ± 0.00a  88.46 ± 4.28c  78.81 ± 2.98c  67.37 ± 3.52d  WHY -4  100.00 ± 0.00a  92.39 ± 2.84b  86.14 ± 3.08b  72.20 ± 4.03c  WHY -5  100.00 ± 0.00a  90.95 ± 3.69b  85.46 ± 4.62b  78.42 ± 2.46b  WHY -6  100.00 ± 0.00a  91.85 ± 3.82b  81.02 ± 3.69b,c  64.65 ± 4.03d  WHY -7  100.00 ± 0.00a  98.90 ± 4.64a  95.90 ± 3.87a  90.30 ± 3.89a  Mean values ± standard deviation. a-dMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at pH 5.5 had been used as a control, measured with 100%. View Large Table 5. Survival percentage (%) of S. cerevisiae strains incubated at low pH for 24 h.   pH  Strains  5.51  4.0  3.0  2.0  WHY -1  100.00 ± 0.00a  93.92 ± 2.78b  84.15 ± 4.23b  72.79 ± 3.54c  WHY -2  100.00 ± 0.00a  98.75 ± 3.05a  95.57 ± 3.16a  90.29 ± 2.71a  WHY -3  100.00 ± 0.00a  88.46 ± 4.28c  78.81 ± 2.98c  67.37 ± 3.52d  WHY -4  100.00 ± 0.00a  92.39 ± 2.84b  86.14 ± 3.08b  72.20 ± 4.03c  WHY -5  100.00 ± 0.00a  90.95 ± 3.69b  85.46 ± 4.62b  78.42 ± 2.46b  WHY -6  100.00 ± 0.00a  91.85 ± 3.82b  81.02 ± 3.69b,c  64.65 ± 4.03d  WHY -7  100.00 ± 0.00a  98.90 ± 4.64a  95.90 ± 3.87a  90.30 ± 3.89a    pH  Strains  5.51  4.0  3.0  2.0  WHY -1  100.00 ± 0.00a  93.92 ± 2.78b  84.15 ± 4.23b  72.79 ± 3.54c  WHY -2  100.00 ± 0.00a  98.75 ± 3.05a  95.57 ± 3.16a  90.29 ± 2.71a  WHY -3  100.00 ± 0.00a  88.46 ± 4.28c  78.81 ± 2.98c  67.37 ± 3.52d  WHY -4  100.00 ± 0.00a  92.39 ± 2.84b  86.14 ± 3.08b  72.20 ± 4.03c  WHY -5  100.00 ± 0.00a  90.95 ± 3.69b  85.46 ± 4.62b  78.42 ± 2.46b  WHY -6  100.00 ± 0.00a  91.85 ± 3.82b  81.02 ± 3.69b,c  64.65 ± 4.03d  WHY -7  100.00 ± 0.00a  98.90 ± 4.64a  95.90 ± 3.87a  90.30 ± 3.89a  Mean values ± standard deviation. a-dMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at pH 5.5 had been used as a control, measured with 100%. View Large Survival at Different Concentrations of Bile Salts The bile salts tolerance of the yeast strains is shown in Table 6. Except WHY-1 and WHY-4, the rest of the strains were able to survive in the presence of 0.30% bile salts. The survival of WHY-2, WHY-6, and WHY-7 was >90% in the presence of 0.60% bile salts. Table 6. Survival percentage (%) of S. cerevisiae strains incubated in the presence of different concentrations of bile salts for 24 h.   Bile salts (%)  Strains  01  0.30  0.60  WHY -1  100.00 ± 0.00a  73.06 ± 2.34b  69.37 ± 2.56c  WHY -2  100.00 ± 0.00a  97.70 ± 3.33a  92.46 ± 2.4a  WHY -3  100.00 ± 0.00a  91.45 ± 4.21a  81.71 ± 3.27b  WHY -4  100.00 ± 0.00a  60.06 ± 3.82b  52.65 ± 4.46d  WHY -5  100.00 ± 0.00a  96.96 ± 2.19a  80.37 ± 3.57b  WHY -6  100.00 ± 0.00a  96.54 ± 4.04a  93.08 ± 3.71a  WHY -7  100.00 ± 0.00a  97.07 ± 3.19a  93.32 ± 3.02a    Bile salts (%)  Strains  01  0.30  0.60  WHY -1  100.00 ± 0.00a  73.06 ± 2.34b  69.37 ± 2.56c  WHY -2  100.00 ± 0.00a  97.70 ± 3.33a  92.46 ± 2.4a  WHY -3  100.00 ± 0.00a  91.45 ± 4.21a  81.71 ± 3.27b  WHY -4  100.00 ± 0.00a  60.06 ± 3.82b  52.65 ± 4.46d  WHY -5  100.00 ± 0.00a  96.96 ± 2.19a  80.37 ± 3.57b  WHY -6  100.00 ± 0.00a  96.54 ± 4.04a  93.08 ± 3.71a  WHY -7  100.00 ± 0.00a  97.07 ± 3.19a  93.32 ± 3.02a  Mean values ± standard deviation. a-dMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at 0% bile salts had been used as a control, measured with 100%. View Large Table 6. Survival percentage (%) of S. cerevisiae strains incubated in the presence of different concentrations of bile salts for 24 h.   Bile salts (%)  Strains  01  0.30  0.60  WHY -1  100.00 ± 0.00a  73.06 ± 2.34b  69.37 ± 2.56c  WHY -2  100.00 ± 0.00a  97.70 ± 3.33a  92.46 ± 2.4a  WHY -3  100.00 ± 0.00a  91.45 ± 4.21a  81.71 ± 3.27b  WHY -4  100.00 ± 0.00a  60.06 ± 3.82b  52.65 ± 4.46d  WHY -5  100.00 ± 0.00a  96.96 ± 2.19a  80.37 ± 3.57b  WHY -6  100.00 ± 0.00a  96.54 ± 4.04a  93.08 ± 3.71a  WHY -7  100.00 ± 0.00a  97.07 ± 3.19a  93.32 ± 3.02a    Bile salts (%)  Strains  01  0.30  0.60  WHY -1  100.00 ± 0.00a  73.06 ± 2.34b  69.37 ± 2.56c  WHY -2  100.00 ± 0.00a  97.70 ± 3.33a  92.46 ± 2.4a  WHY -3  100.00 ± 0.00a  91.45 ± 4.21a  81.71 ± 3.27b  WHY -4  100.00 ± 0.00a  60.06 ± 3.82b  52.65 ± 4.46d  WHY -5  100.00 ± 0.00a  96.96 ± 2.19a  80.37 ± 3.57b  WHY -6  100.00 ± 0.00a  96.54 ± 4.04a  93.08 ± 3.71a  WHY -7  100.00 ± 0.00a  97.07 ± 3.19a  93.32 ± 3.02a  Mean values ± standard deviation. a-dMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at 0% bile salts had been used as a control, measured with 100%. View Large Tolerance to Nutrition Starvation The tolerance of the yeast strains to nutrition starvation is given in Table 7. With the prolongation of starvation time, the CFU of all the strains decreased, with WHY-2 and WHY-7 exhibiting the highest number of CFUs on the 10th day of starvation. Table 7. Tolerance of the S. cerevisiae strains to nutrition starvation (107 CFU/mL).   Time/d  Strains  2  4  6  8  10  WHY -1  9.77 ± 0.04b  4.23 ± 0.03c  3.43 ± 0.03c  3.13 ± 0.02b  2.33 ± 0.01b  WHY -2  11.32 ± 0.03a  10.00 ± 0.01a  9.31 ± 0.05a  6.21 ± 0.04a  4.45 ± 0.02a  WHY -3  9.67 ± 0.05b  8.33 ± 0.14b  7.16 ± 0.04b  5.75 ± 0.03a  2.01 ± 0.01b  WHY -4  9.23 ± 0.06b  5.30 ± 0.05c  4.63 ± 0.03c  2.03 ± 0.02c  1.15 ± 0.02c  WHY -5  9.30 ± 0.10b  8.42 ± 0.06b  7.76 ± 0.05b  3.67 ± 0.04b  1.68 ± 0.04a,b  WHY -6  9.97 ± 0.05b  4.43 ± 0.02c  3.67 ± 0.02c  2.93 ± 0.05b  1.03 ± 0.03c  WHY -7  9.63 ± 0.06b  8.30 ± 0.01b  7.96 ± 0.12b  6.16 ± 0.07a  3.63 ± 0.02a    Time/d  Strains  2  4  6  8  10  WHY -1  9.77 ± 0.04b  4.23 ± 0.03c  3.43 ± 0.03c  3.13 ± 0.02b  2.33 ± 0.01b  WHY -2  11.32 ± 0.03a  10.00 ± 0.01a  9.31 ± 0.05a  6.21 ± 0.04a  4.45 ± 0.02a  WHY -3  9.67 ± 0.05b  8.33 ± 0.14b  7.16 ± 0.04b  5.75 ± 0.03a  2.01 ± 0.01b  WHY -4  9.23 ± 0.06b  5.30 ± 0.05c  4.63 ± 0.03c  2.03 ± 0.02c  1.15 ± 0.02c  WHY -5  9.30 ± 0.10b  8.42 ± 0.06b  7.76 ± 0.05b  3.67 ± 0.04b  1.68 ± 0.04a,b  WHY -6  9.97 ± 0.05b  4.43 ± 0.02c  3.67 ± 0.02c  2.93 ± 0.05b  1.03 ± 0.03c  WHY -7  9.63 ± 0.06b  8.30 ± 0.01b  7.96 ± 0.12b  6.16 ± 0.07a  3.63 ± 0.02a  Mean values ± standard deviation. a-cMeans within the same column lacking a common superscript differ (P < 0.05). View Large Table 7. Tolerance of the S. cerevisiae strains to nutrition starvation (107 CFU/mL).   Time/d  Strains  2  4  6  8  10  WHY -1  9.77 ± 0.04b  4.23 ± 0.03c  3.43 ± 0.03c  3.13 ± 0.02b  2.33 ± 0.01b  WHY -2  11.32 ± 0.03a  10.00 ± 0.01a  9.31 ± 0.05a  6.21 ± 0.04a  4.45 ± 0.02a  WHY -3  9.67 ± 0.05b  8.33 ± 0.14b  7.16 ± 0.04b  5.75 ± 0.03a  2.01 ± 0.01b  WHY -4  9.23 ± 0.06b  5.30 ± 0.05c  4.63 ± 0.03c  2.03 ± 0.02c  1.15 ± 0.02c  WHY -5  9.30 ± 0.10b  8.42 ± 0.06b  7.76 ± 0.05b  3.67 ± 0.04b  1.68 ± 0.04a,b  WHY -6  9.97 ± 0.05b  4.43 ± 0.02c  3.67 ± 0.02c  2.93 ± 0.05b  1.03 ± 0.03c  WHY -7  9.63 ± 0.06b  8.30 ± 0.01b  7.96 ± 0.12b  6.16 ± 0.07a  3.63 ± 0.02a    Time/d  Strains  2  4  6  8  10  WHY -1  9.77 ± 0.04b  4.23 ± 0.03c  3.43 ± 0.03c  3.13 ± 0.02b  2.33 ± 0.01b  WHY -2  11.32 ± 0.03a  10.00 ± 0.01a  9.31 ± 0.05a  6.21 ± 0.04a  4.45 ± 0.02a  WHY -3  9.67 ± 0.05b  8.33 ± 0.14b  7.16 ± 0.04b  5.75 ± 0.03a  2.01 ± 0.01b  WHY -4  9.23 ± 0.06b  5.30 ± 0.05c  4.63 ± 0.03c  2.03 ± 0.02c  1.15 ± 0.02c  WHY -5  9.30 ± 0.10b  8.42 ± 0.06b  7.76 ± 0.05b  3.67 ± 0.04b  1.68 ± 0.04a,b  WHY -6  9.97 ± 0.05b  4.43 ± 0.02c  3.67 ± 0.02c  2.93 ± 0.05b  1.03 ± 0.03c  WHY -7  9.63 ± 0.06b  8.30 ± 0.01b  7.96 ± 0.12b  6.16 ± 0.07a  3.63 ± 0.02a  Mean values ± standard deviation. a-cMeans within the same column lacking a common superscript differ (P < 0.05). View Large Determination of Cell Surface Hydrophobicity The results of cell surface hydrophobicity of the yeast strains are shown in Figure 1. Maximum cell surface hydrophobicity of 73.71% was exhibited by WHY-6, followed by WHY-7 and WHY-2. Figure 1. View largeDownload slide Cell surface hydrophobicity of the S. cerevisiae strains. Error bars indicate standard deviations. Figure 1. View largeDownload slide Cell surface hydrophobicity of the S. cerevisiae strains. Error bars indicate standard deviations. Autoaggregation It can be observed from Figure 2 that the autoaggregation percentage for the seven yeast strains ranged from 96.68% (WHY-5) to 98.99% (WHY-2) at 24 h of incubation. At 2 h of incubation, the autoaggregation percentage for the seven strains presented only slight variation, from 24.06% (WHY-1) to 38.86% (WHY-7). In contrast, at 4 h of incubation, all the yeast strains rapidly and automatically aggregated, and the autoaggregation percentage for WHY-2, WHY-5, and WHY-7 was significantly higher, when compared with that for the other four strains. Figure 2. View largeDownload slide Autoaggregation percentage for the S. cerevisiae strains after 2 h (light gray), 4 h (gray), and 24 h (dark gray) of incubation. Error bars indicate standard deviations. Figure 2. View largeDownload slide Autoaggregation percentage for the S. cerevisiae strains after 2 h (light gray), 4 h (gray), and 24 h (dark gray) of incubation. Error bars indicate standard deviations. DISCUSSION Yeasts are one of the important feed probiotics, which are resistant to changes in intestinal pH and can regulate the micro-ecological balance of the intestinal environment and improve animal immune capacity (Garcia-Hernandez et al., 2012; Tomicic et al., 2016). It is generally believed that the best source of an ideal probiotic strain is the intestine of the native animal (Garcia-Hernandez et al., 2012; Zhang et al., 2013; Forkus et al., 2017; He et al., 2017). Yeasts do not belong to the gut-inherent microbial flora, and occur only sporadically in the gut, playing roles in the digestive tract of animals for survival (Binetti et al., 2013; Tomicic et al., 2016). In the present study, 35 yeast strains were isolated from the digestive tract of Cherry Valley ducks collected from four different regions, which suggested that yeasts can tolerate the intestinal environment of meat ducks, but occur as foreign microflora in the intestine. In China, products containing Saccharomyces spp. have been listed as feed ingredients in Feed Ingredients Catalog (2013), indicating that Saccharomyces spp. used in feed are safe. However, the safe strains that can be used in probiotics must undergo a series of screening. As in vivo studies investigating the health benefits of potential probiotics are time-consuming and often expensive, the consequent use of in vitro tests as selection criteria is unavoidable to reduce the number of strains and determine the most effective microorganism (Valeriano et al., 2014; Iaconelli et al., 2015; Forkus et al., 2017). Resistance to pH and bile salts is of significant importance in the survival and growth of microorganisms in the intestinal tract and a prerequisite for probiotics, and S. cerevisiae is well-tolerant to acidic environment (Garcia-Hernandez et al., 2012; Binetti et al., 2013). In the present study, most of the yeast strains were found to survive at pH 3.0–4.0; however, only WHY-2 and WHY-7 presented higher survival rate at pH 2.0. Furthermore, strains WHY-2, WHY-6, and WHY-7 exhibited higher survival rate in the presence of 0.60% bile salts. As animal body temperature is generally about 37°C, limiting temperature is 42°C, and optimal temperature for yeasts is usually 30°C, heat tolerance screening of candidate yeast strains is necessary. In the present study, the strains WHY-2, WHY-6, and WHY-7 survived well at 37°C, while strains WHY-2 and WHY-7 could even survive at 42°C. Furthermore, as the hind gut of animals is often a nutrient-deficit environment, nutrition starvation screening of yeast strains is also vital. The results of the present study showed that the cell counts of all the yeast strains were reduced owing to starvation. However, the strains WHY-2 and WHY-7 exhibited relatively higher cell counts on the 10th day of starvation. Cell surface hydrophobicity is one of the important properties of probiotics, and has been primarily studied based on microbial adhesion to hydrocarbons (Gil-Rodríguez et al., 2015). In the present study, the selected isolates presented strong hydrophobicity in n-hexadecane, indicating potential putative probiotic property (Ogunremi et al., 2015). In particular, the strains WHY-6, WHY-2, and WHY-7 showed higher cell surface hydrophobicity. Another characteristic of a potential probiotic microorganism is the ability to form cellular aggregates, because aggregates can increase microbial adherence to the intestine, thus providing advantages in colonization of the GI tract (García-Cayuela et al., 2014). As yeast cells are bigger and heavier than bacteria, they precipitate quickly and in higher proportion (Gil-Rodríguez et al., 2015). In the present study, the autoaggregation percentages for all the strains were not high at 2 h, but remarkably increased at 4 h of incubation. These results are similar to those reported by Gil-Rodríguez et al. (2015), who demonstrated that the yeasts displayed rapid autoaggregation within the first 4 h of incubation. CONCLUSION In the present study, 35 yeast strains were isolated and identified from the GI tract of ducks, of which seven strains belonged to S. cerevisiae. These seven yeast strains were screened for their cell surface hydrophobicity, autoaggregation ability, and tolerance to high temperature, low pH, bile salts, and nutrition starvation. The results obtained showed that strains WHY-2 and WHY-7 had higher performance, and could be potential probiotic candidates. Thus, the findings of this study could help in selecting probiotic yeast candidates for use in poultry industry. Acknowledgements This work was funded by the Wuhan Science and Technology Bureau, Wuhan city, China (Project 2014020101010073). We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript. Notes The appropriate scientific section for the paper is “Microbiology and Food Safety”. REFERENCES Binetti A., Carrasco M., Reinheimer J., Suarez V.. 2013. 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Yeast  14: 77– 100. Google Scholar CrossRef Search ADS PubMed  Zhang W., Liu M., Dai X.. 2013. Biological characteristics and probiotic effect of Leuconostoc lactis strain isolated from the intestine of black porgy fish. Braz. J. Microbiol.  44: 685– 691. Google Scholar CrossRef Search ADS PubMed  © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Identification and characterization of probiotic yeast isolated from digestive tract of ducks

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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10.3382/ps/pey152
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Abstract

ABSTRACT The objective of this study was to isolate and identify yeast strains from the digestive tract of ducks, and evaluate in vitro their potential as probiotics in poultry. The yeast strains were isolated using malt extract agar medium, and identified through morphological, physiological, and biochemical tests as well as sequence homology analyses of 26S rDNA D1/D2 region. A total of 35 yeast strains were isolated from the guts of Cherry Valley meat ducks, including seven strains of Saccharomyces cerevisiae (S. cerevisiae). These seven strains of S. cerevisiae were further screened for their use as alternative yeast probiotics strains for poultry feed. The yeast strains were characterized for their cell surface hydrophobicity, autoaggregation ability, and resistance to high temperature (30°C, 37°C, and 42°C), low pH (2.0, 3.0, and 4.0), bile salts (0.3% and 0.6%), and nutrition starvation (2, 4, 6, 8, 10, and 12 days). The isolates of WHY-2 and WHY-7 had a higher survival percentage at 37°C, pH 2.0, 0.60% poultry bile salts, and 10 days of nutrition starvation, with higher cell surface hydrophobicity and autoaggregation, when compared with the other isolates, suggesting that the isolates WHY-2 and WHY-7, could be used as probiotic candidates. The data obtained in this study could help in selecting probiotic yeast candidates for use in poultry industry. INTRODUCTION Growth-promoting antimicrobials, such as ionophore antibiotics, have been widely utilized and are still being used in some countries. However, owing to the increasing safety concerns about the risk of development of antibiotics resistance and persistence of chemical residues in animal products, some strategies based on supplementation of more “natural” products such as probiotics, have been established to improve herd health and productivity (Francisco et al., 2009). Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2006). Several criteria are required for a microorganism to be considered as a probiotic: it must be innocuous, causing neither toxicity nor illness to the host, it must occur in high amounts in the product with which it is administered and maintain its viability during the product's shelf-life, and it must be technologically usable and survive gastrointestinal (GI) transit (Hill et al., 2014; Larsen et al., 2014). Yeasts are one of the major groups of probiotics (Czerucka and Rampal, 2002). In the past decades, yeasts have been considered as one of the microorganisms that could be used in probiotics for poultry (Fleet, 2007; Jacques and Casaregola, 2008). Yeasts have unique properties, such as not being affected by antibacterial treatments, which can help to alleviate antibiotic-associated diarrhea (Moslehijenabian et al., 2010; Hatoum et al., 2012). Currently, the conventional yeast Saccharomyces cerevisiae is the most common animal probiotic yeast available on the market (Czerucka et al., 2007; Didari et al., 2014). However, the effects of different yeast strains on animals are significantly different. It is generally believed that strains isolated from the animal digestive ecosystem are adapted to the GI tract environment and could be used as ideal probiotics (Hume et al., 2012). Nevertheless, each strain must be well identified and characterized in vitro and in vivo (FAO/WHO, 2006) before being used for these purposes. The in vitro selection criteria for probiotic candidates include tolerance to GI tract conditions and ability of the candidate strains to adhere to intestinal epithelial cells (Trotman, 2002; Van et al., 2005). To reach the target site of action alive, the potential probiotic microorganisms must be able to tolerate a variety of adverse conditions of the GI tract, such as temperature stress (i.e., internal body temperature of 37°C), acidic gastric juice, bile salts, and nutrition starvation (Kumura et al., 2004; Van et al., 2005). Hence, selection of probiotic microorganisms is typically performed in vitro by screening the probiotic candidates for survival under simulated GI tract conditions (Martins et al., 2008). Cell-surface hydrophobicity and autoaggregation are important physicochemical forces that help in the adherence of cells to biological and abiotic surfaces, and may be the main factors determining the adhesion properties of cells, which may also be the basis for probiotics to function as a biological barrier (Van et al., 2005). Thus, the objective of the present study was to isolate, identify, and screen (in vitro) yeast strains (S. cerevisiae) from duck GI tracts for their probiotic potential in the poultry industry. MATERIALS AND METHODS Yeast Isolation Procedures A total of 24 healthy ducks were obtained from duck farm and farmers’ market in Yichang and Wuhan, respectively, and yeast strains were isolated from these ducks’ GI tracts. In brief, the ducks’ digestive tract contents were extracted. Each sample was diluted 10-fold in sterile saline (0.85% NaCl) and incubated at ambient temperature for 90 min. Serial dilutions of each sample were performed and 1 mL aliquots of appropriate dilutions were inoculated (in duplicates) by the pour-plate method on yeast peptone dextrose (YPD) agar plates (containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 20 g/L agar) supplemented with chloramphenicol (200 mg/L) and incubated at 30°C for 48 h (Kurtzman, 2011). Subsequently, colonies showing different morphologies were selected and inoculated thrice on malt extract agar plates. The pure cultures were preserved on Sabouraud agar plates (Scharlau, Spain) at 8 ± 2°C until further use. Characterization of Yeast Strains All the yeasts were preliminarily grouped based on their cultural morphology, urease production, and physiological characteristics such as carbon assimilation, nitrogen sources, and amyloid compounds production (Yarrow, 1998). The yeast strains were cultured in YPD broth for 24 h at 30°C. The total genomic DNA of the yeast strains were extracted and purified by using a yeast genomic DNA extraction kit (Tiangen Biotech Co. Ltd, Beijing, China). The D1/D2 region of the large subunit of 26S rDNA of the selected yeast strains was amplified with universal primers, 5′-GCATATCAATAAGCGGAGGAAAAG-3′ (forward) and 5′-GGTCCGTGTTTCAAGACGG-3′ (reverse) (Kurtzman and Robnett, 1998). The primers were synthesized by Sangon Biotech Co. Ltd, Shanghai, China. PCR was performed in a 50 μL reaction system consisting of 25 μL of 2 × Taq PCR Master Mix (Tiangen Biotech Co. Ltd), 4 μL of DNA template, 1 μL of each primer, and 19 μL of sterile deionized H2O. The reaction conditions were as follows: initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 45 s, renaturation at 55°C for 1 min, and extension at 72°C for 70 s, and final extension at 72°C for 10 min. The purity of the PCR products was observed with 1% agarose gel electrophoresis. The sequencing of the 26S rDNA gene was performed by Sangon Biotech Co. Ltd. The yeast strains were identified after aligning the sequences to a nonredundant database (www.ncbi.nlm.nih.gov/blast). The strains of S. cerevisiae were identified for further investigation as candidate probiotics. Tolerance to High Temperature Sterile YPD broths were inoculated with 1% (v/v) 18 h culture broth of the test isolate and incubated at 30°C, 37°C, or 42°C. After 30 min, samples were collected and the viable cells counts (CFU/mL) were determined (Garcıa-Hernandez et al., 2012). The assay was performed in triplicate and the survival percentage (S) was calculated as follows:   \[ {\rm{S}}(\%) = \left( {\rm{N}}_{\rm{t}} \times 100 \right)/{{\rm{N}}_0} \] where Nt is CFU/mL at temperature t = 37°C or 42°C, and N0 is CFU/mL at 30°C. Tolerance to Low pH A suspension of the test isolate was prepared in YPD broth and its optical density at 600 nm (OD600) was adjusted to 1.0. Then, the suspension was centrifuged at 5000 × g for 10 min, washed with buffered phosphate solution (10 mM, pH 7.4), and resuspended in 3 mL of buffered solution phosphate solution with pH adjusted to 2.0, 3.0, or 4.0 with 90% lactic acid. After 24 h of incubation, the samples were serially diluted and inoculated onto YPD agar plates to determine the cells counts. The experiment was performed in triplicate according to completely randomized design, and the survival percentage (S) was calculated as follows:   \[ {\rm{S}}(\%) = {\rm{[(CFU/mL)}}_{{\rm{YPD }} + {\rm{Lactic\,\,acid}}} \times 100]/{{\rm{(CFU/mL)}}_{{\rm{YPD}}}}\] Tolerance to Bile Salts A suspension of the test isolate was prepared in YPD broth and its OD600 was adjusted to 1.0. Then, the suspension was serially diluted, inoculated onto Sabouraud agar plates containing 0%, 0.30%, or 0.60% (w/v) poultry bile salts (BoMei Biotech Co. Ltd, Shanghai, China), incubated at 30°C for 72 h, and the cell counts (CFU/mL) for each treatment were determined. The assay was performed in triplicate and the survival percentage (S) was calculated as follows:   \[ {\rm{S}}(\%) = [\rm{(CFU/mL)}_{\rm YPD+Salts} \times 100] /{{\rm (CFU/mL)}_{\rm YPD} } \] Tolerance to Nutrition Starvation A suspension of the test isolate was prepared in YPD broth and its OD600 was adjusted to 1.0. Then, 1% (v/v) suspension was inoculated into sterile water. After 2, 4, 6, 8, 10, or 12 days of incubation at 30°C, the suspensions were re-inoculated into YPD broth and incubated at 30°C for 72 h for growth experiment. The cell counts (CFU/mL) for each treatment were determined. Hydrophobicity of the Cell Surface The surface hydrophobicity of the S. cerevisiae strains was determined based on the adhesive capability of the yeast strains to hydrocarbon compounds (Holle et al., 2012). The yeast cells (5 × 106 cells/mL) were suspended in phosphate buffer (pH 7.1) containing 0.2 g/L MgSO4 and 1.8 g/L urea. Then, the cell suspension (10.0 mL) was placed in standard glass test tubes and acid-washed. Subsequently, n-hexadecane (2 mL) was added to each tube, vortex-mixed for 2 min, and left to stand for 15 min to ensure complete separation of the two phases. The aqueous phase was carefully removed and its absorbance was determined at 600 nm. The hydrophobicity of the cell surface (%) was calculated according to the following equation:   \[ {\rm{Hydrophobicity}}(\%) = \left( {{\rm{1}} - {\rm{A/}}{{\rm{A}}_{\rm{0}}}} \right) \times {\rm{100}} \] where A0 is the initial absorbance of the cell suspension, and A is the absorbance of the aqueous phase after mixing. Autoaggregation To measure the autoaggregation percentage, 80 mL of the YPD culture broth of the test isolate were harvested by centrifugation (4000 × g, 5 min) and washed twice with a sterile solution of 0.9% NaCl. The resulting pellet was resuspended in 5 mL of 0.9% NaCl solution and transferred to a disposable plastic cuvette. Subsequently, the OD600 of the suspension was measured at 0, 2, 4, and 24 h. The autoaggregation percentage was calculated as follows:   \[ {\rm{Autoaggregation }}(\%) = \left( {{\rm{1}} - {\rm{A/}}{{\rm{A}}_{\rm{0}}}} \right) \times {\rm{100}} \] where At represents the OD at time t = 2, 4, or 24 h, and A0 represents the OD at t = 0 h. Statistical Analysis The data obtained were subjected to analysis of variance and the significant differences were compared using Duncan's multiple range test. Values of P < 0.05 indicated statistically significant differences. The data analyses were performed with SPSS software (ver 22.0; IBM, Armonk, NY, USA). All the results are presented as the mean with standard deviation of triplicate values. RESULTS Isolation and Identification of Yeast Strains A total of 35 yeast colonies with different macroscopic morphologies were isolated from duck GI tracts. After comparing the sequences of D1/D2 region of 26S rDNA extracted from the yeast strains with those in the GenBank database, the strains were identified as Candida albicans (Y-1, Y-2, Y-3, Y-6, Y-9, Y-10, Y-15, Y-16, Y-21, Y-30), Cryptococcus arboriformis (Y-4, Y-22), Kazachstania bovina (Y-5, Y-7, Y-17, Y-23), Candida parapsilosis (Y-8, Y-11, Y-14, Y-24, Y-26, Y-27, Y-28), Lodderomyces elongisporus (Y-12, Y-35), Kodamaea ohmeri (Y-13), S. cerevisiae (Y-18, Y-19, Y-20, Y-25, Y-31, Y-33, Y-34), and Candida glabrata (Y-29, Y-32) (Table 1). As S. cerevisiae is the most common animal probiotic yeast, the seven strains of S. cerevisiae were chosen for further investigation and renamed as WHY-1, WHY-2, WHY-3, WHY-4, WHY-5, WHY-6, and WHY-7. The sequences of the D1/D2 region of 26S rDNA of these seven strains have been submitted to GenBank (Table 2). The biochemical identification results showed that all the S. cerevisiae strains were able to ferment D-glucose, maltose, and sucrose, but not lactose. Furthermore, all the strains, except WHY-4, could ferment galactose. With regard to melibiose fermentation, except WHY-2 and WHY-6, the rest of the strains showed positive results. Moreover, while none of the strains could assimilate potassium nitrate, L-lysine, and cadaverine, they could grow in non-niacin YPD agar. In addition, WHY-2, WHY-3, and WHY-6 were positive in the non-pyridoxine test, whereas WHY-2, WHY-4, and WHY-5 were positive in the non-thiamine test. With regard to 0.01% cycloheximide tests, starch-like compound formation test, urea decomposition test, and 50% D-glucose growth test, the results were negative for all the seven strains (Table 3). Table 1. Molecular identification of the yeast strains isolated from duck gastrointestinal tracts by sequencing of the D1/D2 region of 26S rDNA gene. Strains  Molecular identification  Similarity (%)  GenBank ID of type strain  Y-1, 2, 3, 6, 9, 10, 15, 16, 21, 30  Candida albicans  99.0  KJ534502  Y-4, 22  Cryptococcus arboriformis  99.0  AB260936  Y-5, 7, 17, 23  Kazachstania bovina  99.5  AB499993  Y-8, 11, 14, 24, 26, 27, 28  Candida parapsilosis  99.0  FJ746059  Y-12, 35  Lodderomyces elongisporus  100.0  LC195009  Y-13  Kodamaea ohmeri  99.0  GU597323  Y-18, 19, 20, 25, 31, 33, 34  Saccharomyces cerevisiae  99.6  KM655848  Y-29, 32  Candida glabrata  99.5  HM627130  Strains  Molecular identification  Similarity (%)  GenBank ID of type strain  Y-1, 2, 3, 6, 9, 10, 15, 16, 21, 30  Candida albicans  99.0  KJ534502  Y-4, 22  Cryptococcus arboriformis  99.0  AB260936  Y-5, 7, 17, 23  Kazachstania bovina  99.5  AB499993  Y-8, 11, 14, 24, 26, 27, 28  Candida parapsilosis  99.0  FJ746059  Y-12, 35  Lodderomyces elongisporus  100.0  LC195009  Y-13  Kodamaea ohmeri  99.0  GU597323  Y-18, 19, 20, 25, 31, 33, 34  Saccharomyces cerevisiae  99.6  KM655848  Y-29, 32  Candida glabrata  99.5  HM627130  View Large Table 1. Molecular identification of the yeast strains isolated from duck gastrointestinal tracts by sequencing of the D1/D2 region of 26S rDNA gene. Strains  Molecular identification  Similarity (%)  GenBank ID of type strain  Y-1, 2, 3, 6, 9, 10, 15, 16, 21, 30  Candida albicans  99.0  KJ534502  Y-4, 22  Cryptococcus arboriformis  99.0  AB260936  Y-5, 7, 17, 23  Kazachstania bovina  99.5  AB499993  Y-8, 11, 14, 24, 26, 27, 28  Candida parapsilosis  99.0  FJ746059  Y-12, 35  Lodderomyces elongisporus  100.0  LC195009  Y-13  Kodamaea ohmeri  99.0  GU597323  Y-18, 19, 20, 25, 31, 33, 34  Saccharomyces cerevisiae  99.6  KM655848  Y-29, 32  Candida glabrata  99.5  HM627130  Strains  Molecular identification  Similarity (%)  GenBank ID of type strain  Y-1, 2, 3, 6, 9, 10, 15, 16, 21, 30  Candida albicans  99.0  KJ534502  Y-4, 22  Cryptococcus arboriformis  99.0  AB260936  Y-5, 7, 17, 23  Kazachstania bovina  99.5  AB499993  Y-8, 11, 14, 24, 26, 27, 28  Candida parapsilosis  99.0  FJ746059  Y-12, 35  Lodderomyces elongisporus  100.0  LC195009  Y-13  Kodamaea ohmeri  99.0  GU597323  Y-18, 19, 20, 25, 31, 33, 34  Saccharomyces cerevisiae  99.6  KM655848  Y-29, 32  Candida glabrata  99.5  HM627130  View Large Table 2. GenBank accession numbers for the D1/D2 region of 26S rDNA of the S. cerevisiae strains from duck gastrointestinal tracts. Strains  Accession #  WHY -1  MG641146  WHY -2  MG641147  WHY -3  MG641148  WHY -4  MG641149  WHY -5  MG641150  WHY -6  MG641151  WHY -7  MG641152  Strains  Accession #  WHY -1  MG641146  WHY -2  MG641147  WHY -3  MG641148  WHY -4  MG641149  WHY -5  MG641150  WHY -6  MG641151  WHY -7  MG641152  View Large Table 2. GenBank accession numbers for the D1/D2 region of 26S rDNA of the S. cerevisiae strains from duck gastrointestinal tracts. Strains  Accession #  WHY -1  MG641146  WHY -2  MG641147  WHY -3  MG641148  WHY -4  MG641149  WHY -5  MG641150  WHY -6  MG641151  WHY -7  MG641152  Strains  Accession #  WHY -1  MG641146  WHY -2  MG641147  WHY -3  MG641148  WHY -4  MG641149  WHY -5  MG641150  WHY -6  MG641151  WHY -7  MG641152  View Large Table 3. Biochemical identification of the S. cerevisiae strains from duck gastrointestinal tracts.   Yeast isolates, WHY  Substrate  1  2  3  4  5  6  7  D-Glucose  +  +  +  +  +  +  +  Maltose  +  +  +  +  +  +  +  Galactose  +  +  +  −  +  +  +  Lactose  −  −  −  −  −  −  −  Melibiose  +  −  +  +  +  −  +  Saccharose  +  +  +  +  +  +  +  Niter  −  −  −  −  −  −  −  L-Lysine  −  −  −  −  −  −  −  Cadaverine  −  −  −  −  −  −  −  Non-niacin  +  +  +  +  +  +  +  Non-pyridoxine  −  +  +  −  −  +  −  Non-thiamine  −  +  −  +  +  −  −  0.01% Cycloheximide  −  −  −  −  −  −  −  starch-like compound formation  −  −  −  −  −  −  −  urea decomposition  −  −  −  −  −  −  −  50% D-glucose growth  −  −  −  −  −  −  −  Hypha or pseudohypha  −  +  −  +  −  +  +    Yeast isolates, WHY  Substrate  1  2  3  4  5  6  7  D-Glucose  +  +  +  +  +  +  +  Maltose  +  +  +  +  +  +  +  Galactose  +  +  +  −  +  +  +  Lactose  −  −  −  −  −  −  −  Melibiose  +  −  +  +  +  −  +  Saccharose  +  +  +  +  +  +  +  Niter  −  −  −  −  −  −  −  L-Lysine  −  −  −  −  −  −  −  Cadaverine  −  −  −  −  −  −  −  Non-niacin  +  +  +  +  +  +  +  Non-pyridoxine  −  +  +  −  −  +  −  Non-thiamine  −  +  −  +  +  −  −  0.01% Cycloheximide  −  −  −  −  −  −  −  starch-like compound formation  −  −  −  −  −  −  −  urea decomposition  −  −  −  −  −  −  −  50% D-glucose growth  −  −  −  −  −  −  −  Hypha or pseudohypha  −  +  −  +  −  +  +  +: positive; −: negative. View Large Table 3. Biochemical identification of the S. cerevisiae strains from duck gastrointestinal tracts.   Yeast isolates, WHY  Substrate  1  2  3  4  5  6  7  D-Glucose  +  +  +  +  +  +  +  Maltose  +  +  +  +  +  +  +  Galactose  +  +  +  −  +  +  +  Lactose  −  −  −  −  −  −  −  Melibiose  +  −  +  +  +  −  +  Saccharose  +  +  +  +  +  +  +  Niter  −  −  −  −  −  −  −  L-Lysine  −  −  −  −  −  −  −  Cadaverine  −  −  −  −  −  −  −  Non-niacin  +  +  +  +  +  +  +  Non-pyridoxine  −  +  +  −  −  +  −  Non-thiamine  −  +  −  +  +  −  −  0.01% Cycloheximide  −  −  −  −  −  −  −  starch-like compound formation  −  −  −  −  −  −  −  urea decomposition  −  −  −  −  −  −  −  50% D-glucose growth  −  −  −  −  −  −  −  Hypha or pseudohypha  −  +  −  +  −  +  +    Yeast isolates, WHY  Substrate  1  2  3  4  5  6  7  D-Glucose  +  +  +  +  +  +  +  Maltose  +  +  +  +  +  +  +  Galactose  +  +  +  −  +  +  +  Lactose  −  −  −  −  −  −  −  Melibiose  +  −  +  +  +  −  +  Saccharose  +  +  +  +  +  +  +  Niter  −  −  −  −  −  −  −  L-Lysine  −  −  −  −  −  −  −  Cadaverine  −  −  −  −  −  −  −  Non-niacin  +  +  +  +  +  +  +  Non-pyridoxine  −  +  +  −  −  +  −  Non-thiamine  −  +  −  +  +  −  −  0.01% Cycloheximide  −  −  −  −  −  −  −  starch-like compound formation  −  −  −  −  −  −  −  urea decomposition  −  −  −  −  −  −  −  50% D-glucose growth  −  −  −  −  −  −  −  Hypha or pseudohypha  −  +  −  +  −  +  +  +: positive; −: negative. View Large High Temperature Tolerance The high temperature tolerance of the yeast strains is presented in Table 4. At 37°C, the survival of strains WHY-2, WHY-6, and WHY-7 was significantly higher, when compared with that of the other four strains. Furthermore, at 42°C, the survival of WHY-2 and WHY-7 was significantly higher, when compared with that of the rest of the strains, indicating that WHY-2 and WHY-7 exhibited high temperature tolerance (at both 37°C and 42°C). Table 4. Survival percentage (%) of S. cerevisiae strains incubated at 30°C, 37°C, 42°C, and 48°C for 30 min.   Temperature    Strains  30°C1  37°C  42°C  48°C  WHY -1  100.00 ± 0.00a  67.98 ± 3.51c  57.30 ± 2.37e  3.93 ± 0.97c  WHY -2  100.00 ± 0.00a  95.37 ± 3.20a  90.74 ± 3.20a,b  9.25 ± 0.61a  WHY -3  100.00 ± 0.00a  70.62 ± 1.95c  86.44 ± 4.08b,c  5.65 ± 0.98b,c  WHY -4  100.00 ± 0.00a  80.29 ± 3.34b  75.18 ± 3.32d  7.30 ± 0.26a,b  WHY -5  100.00 ± 0.00a  72.77 ± 4.30b,c  71.96 ± 3.83d  7.59 ± 0.77a,b  WHY -6  100.00 ± 0.00a  91.60 ± 5.25a  80.67 ± 3.36c,d  5.88 ± 0.45b,c  WHY -7  100.00 ± 0.00a  98.04 ± 5.39a  93.14 ± 4.12a  7.84 ± 0.70a,b    Temperature    Strains  30°C1  37°C  42°C  48°C  WHY -1  100.00 ± 0.00a  67.98 ± 3.51c  57.30 ± 2.37e  3.93 ± 0.97c  WHY -2  100.00 ± 0.00a  95.37 ± 3.20a  90.74 ± 3.20a,b  9.25 ± 0.61a  WHY -3  100.00 ± 0.00a  70.62 ± 1.95c  86.44 ± 4.08b,c  5.65 ± 0.98b,c  WHY -4  100.00 ± 0.00a  80.29 ± 3.34b  75.18 ± 3.32d  7.30 ± 0.26a,b  WHY -5  100.00 ± 0.00a  72.77 ± 4.30b,c  71.96 ± 3.83d  7.59 ± 0.77a,b  WHY -6  100.00 ± 0.00a  91.60 ± 5.25a  80.67 ± 3.36c,d  5.88 ± 0.45b,c  WHY -7  100.00 ± 0.00a  98.04 ± 5.39a  93.14 ± 4.12a  7.84 ± 0.70a,b  Mean values ± standard deviation. a-eMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at 30°C had been used as a control, measured with 100%. View Large Table 4. Survival percentage (%) of S. cerevisiae strains incubated at 30°C, 37°C, 42°C, and 48°C for 30 min.   Temperature    Strains  30°C1  37°C  42°C  48°C  WHY -1  100.00 ± 0.00a  67.98 ± 3.51c  57.30 ± 2.37e  3.93 ± 0.97c  WHY -2  100.00 ± 0.00a  95.37 ± 3.20a  90.74 ± 3.20a,b  9.25 ± 0.61a  WHY -3  100.00 ± 0.00a  70.62 ± 1.95c  86.44 ± 4.08b,c  5.65 ± 0.98b,c  WHY -4  100.00 ± 0.00a  80.29 ± 3.34b  75.18 ± 3.32d  7.30 ± 0.26a,b  WHY -5  100.00 ± 0.00a  72.77 ± 4.30b,c  71.96 ± 3.83d  7.59 ± 0.77a,b  WHY -6  100.00 ± 0.00a  91.60 ± 5.25a  80.67 ± 3.36c,d  5.88 ± 0.45b,c  WHY -7  100.00 ± 0.00a  98.04 ± 5.39a  93.14 ± 4.12a  7.84 ± 0.70a,b    Temperature    Strains  30°C1  37°C  42°C  48°C  WHY -1  100.00 ± 0.00a  67.98 ± 3.51c  57.30 ± 2.37e  3.93 ± 0.97c  WHY -2  100.00 ± 0.00a  95.37 ± 3.20a  90.74 ± 3.20a,b  9.25 ± 0.61a  WHY -3  100.00 ± 0.00a  70.62 ± 1.95c  86.44 ± 4.08b,c  5.65 ± 0.98b,c  WHY -4  100.00 ± 0.00a  80.29 ± 3.34b  75.18 ± 3.32d  7.30 ± 0.26a,b  WHY -5  100.00 ± 0.00a  72.77 ± 4.30b,c  71.96 ± 3.83d  7.59 ± 0.77a,b  WHY -6  100.00 ± 0.00a  91.60 ± 5.25a  80.67 ± 3.36c,d  5.88 ± 0.45b,c  WHY -7  100.00 ± 0.00a  98.04 ± 5.39a  93.14 ± 4.12a  7.84 ± 0.70a,b  Mean values ± standard deviation. a-eMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at 30°C had been used as a control, measured with 100%. View Large Survival at Low pH Table 5 shows the survival of the yeast strains at pH 5.5, 4.0, 3.0, and 2.0. Strains WHY-2 and WHY-7 exhibited higher survival at low pH, when compared with the rest of the strains. The survival of WHY-2 and WHY-7 was >90% at pH 2.0, >95% at pH 3.0, and >98% at pH 4.0. Table 5. Survival percentage (%) of S. cerevisiae strains incubated at low pH for 24 h.   pH  Strains  5.51  4.0  3.0  2.0  WHY -1  100.00 ± 0.00a  93.92 ± 2.78b  84.15 ± 4.23b  72.79 ± 3.54c  WHY -2  100.00 ± 0.00a  98.75 ± 3.05a  95.57 ± 3.16a  90.29 ± 2.71a  WHY -3  100.00 ± 0.00a  88.46 ± 4.28c  78.81 ± 2.98c  67.37 ± 3.52d  WHY -4  100.00 ± 0.00a  92.39 ± 2.84b  86.14 ± 3.08b  72.20 ± 4.03c  WHY -5  100.00 ± 0.00a  90.95 ± 3.69b  85.46 ± 4.62b  78.42 ± 2.46b  WHY -6  100.00 ± 0.00a  91.85 ± 3.82b  81.02 ± 3.69b,c  64.65 ± 4.03d  WHY -7  100.00 ± 0.00a  98.90 ± 4.64a  95.90 ± 3.87a  90.30 ± 3.89a    pH  Strains  5.51  4.0  3.0  2.0  WHY -1  100.00 ± 0.00a  93.92 ± 2.78b  84.15 ± 4.23b  72.79 ± 3.54c  WHY -2  100.00 ± 0.00a  98.75 ± 3.05a  95.57 ± 3.16a  90.29 ± 2.71a  WHY -3  100.00 ± 0.00a  88.46 ± 4.28c  78.81 ± 2.98c  67.37 ± 3.52d  WHY -4  100.00 ± 0.00a  92.39 ± 2.84b  86.14 ± 3.08b  72.20 ± 4.03c  WHY -5  100.00 ± 0.00a  90.95 ± 3.69b  85.46 ± 4.62b  78.42 ± 2.46b  WHY -6  100.00 ± 0.00a  91.85 ± 3.82b  81.02 ± 3.69b,c  64.65 ± 4.03d  WHY -7  100.00 ± 0.00a  98.90 ± 4.64a  95.90 ± 3.87a  90.30 ± 3.89a  Mean values ± standard deviation. a-dMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at pH 5.5 had been used as a control, measured with 100%. View Large Table 5. Survival percentage (%) of S. cerevisiae strains incubated at low pH for 24 h.   pH  Strains  5.51  4.0  3.0  2.0  WHY -1  100.00 ± 0.00a  93.92 ± 2.78b  84.15 ± 4.23b  72.79 ± 3.54c  WHY -2  100.00 ± 0.00a  98.75 ± 3.05a  95.57 ± 3.16a  90.29 ± 2.71a  WHY -3  100.00 ± 0.00a  88.46 ± 4.28c  78.81 ± 2.98c  67.37 ± 3.52d  WHY -4  100.00 ± 0.00a  92.39 ± 2.84b  86.14 ± 3.08b  72.20 ± 4.03c  WHY -5  100.00 ± 0.00a  90.95 ± 3.69b  85.46 ± 4.62b  78.42 ± 2.46b  WHY -6  100.00 ± 0.00a  91.85 ± 3.82b  81.02 ± 3.69b,c  64.65 ± 4.03d  WHY -7  100.00 ± 0.00a  98.90 ± 4.64a  95.90 ± 3.87a  90.30 ± 3.89a    pH  Strains  5.51  4.0  3.0  2.0  WHY -1  100.00 ± 0.00a  93.92 ± 2.78b  84.15 ± 4.23b  72.79 ± 3.54c  WHY -2  100.00 ± 0.00a  98.75 ± 3.05a  95.57 ± 3.16a  90.29 ± 2.71a  WHY -3  100.00 ± 0.00a  88.46 ± 4.28c  78.81 ± 2.98c  67.37 ± 3.52d  WHY -4  100.00 ± 0.00a  92.39 ± 2.84b  86.14 ± 3.08b  72.20 ± 4.03c  WHY -5  100.00 ± 0.00a  90.95 ± 3.69b  85.46 ± 4.62b  78.42 ± 2.46b  WHY -6  100.00 ± 0.00a  91.85 ± 3.82b  81.02 ± 3.69b,c  64.65 ± 4.03d  WHY -7  100.00 ± 0.00a  98.90 ± 4.64a  95.90 ± 3.87a  90.30 ± 3.89a  Mean values ± standard deviation. a-dMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at pH 5.5 had been used as a control, measured with 100%. View Large Survival at Different Concentrations of Bile Salts The bile salts tolerance of the yeast strains is shown in Table 6. Except WHY-1 and WHY-4, the rest of the strains were able to survive in the presence of 0.30% bile salts. The survival of WHY-2, WHY-6, and WHY-7 was >90% in the presence of 0.60% bile salts. Table 6. Survival percentage (%) of S. cerevisiae strains incubated in the presence of different concentrations of bile salts for 24 h.   Bile salts (%)  Strains  01  0.30  0.60  WHY -1  100.00 ± 0.00a  73.06 ± 2.34b  69.37 ± 2.56c  WHY -2  100.00 ± 0.00a  97.70 ± 3.33a  92.46 ± 2.4a  WHY -3  100.00 ± 0.00a  91.45 ± 4.21a  81.71 ± 3.27b  WHY -4  100.00 ± 0.00a  60.06 ± 3.82b  52.65 ± 4.46d  WHY -5  100.00 ± 0.00a  96.96 ± 2.19a  80.37 ± 3.57b  WHY -6  100.00 ± 0.00a  96.54 ± 4.04a  93.08 ± 3.71a  WHY -7  100.00 ± 0.00a  97.07 ± 3.19a  93.32 ± 3.02a    Bile salts (%)  Strains  01  0.30  0.60  WHY -1  100.00 ± 0.00a  73.06 ± 2.34b  69.37 ± 2.56c  WHY -2  100.00 ± 0.00a  97.70 ± 3.33a  92.46 ± 2.4a  WHY -3  100.00 ± 0.00a  91.45 ± 4.21a  81.71 ± 3.27b  WHY -4  100.00 ± 0.00a  60.06 ± 3.82b  52.65 ± 4.46d  WHY -5  100.00 ± 0.00a  96.96 ± 2.19a  80.37 ± 3.57b  WHY -6  100.00 ± 0.00a  96.54 ± 4.04a  93.08 ± 3.71a  WHY -7  100.00 ± 0.00a  97.07 ± 3.19a  93.32 ± 3.02a  Mean values ± standard deviation. a-dMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at 0% bile salts had been used as a control, measured with 100%. View Large Table 6. Survival percentage (%) of S. cerevisiae strains incubated in the presence of different concentrations of bile salts for 24 h.   Bile salts (%)  Strains  01  0.30  0.60  WHY -1  100.00 ± 0.00a  73.06 ± 2.34b  69.37 ± 2.56c  WHY -2  100.00 ± 0.00a  97.70 ± 3.33a  92.46 ± 2.4a  WHY -3  100.00 ± 0.00a  91.45 ± 4.21a  81.71 ± 3.27b  WHY -4  100.00 ± 0.00a  60.06 ± 3.82b  52.65 ± 4.46d  WHY -5  100.00 ± 0.00a  96.96 ± 2.19a  80.37 ± 3.57b  WHY -6  100.00 ± 0.00a  96.54 ± 4.04a  93.08 ± 3.71a  WHY -7  100.00 ± 0.00a  97.07 ± 3.19a  93.32 ± 3.02a    Bile salts (%)  Strains  01  0.30  0.60  WHY -1  100.00 ± 0.00a  73.06 ± 2.34b  69.37 ± 2.56c  WHY -2  100.00 ± 0.00a  97.70 ± 3.33a  92.46 ± 2.4a  WHY -3  100.00 ± 0.00a  91.45 ± 4.21a  81.71 ± 3.27b  WHY -4  100.00 ± 0.00a  60.06 ± 3.82b  52.65 ± 4.46d  WHY -5  100.00 ± 0.00a  96.96 ± 2.19a  80.37 ± 3.57b  WHY -6  100.00 ± 0.00a  96.54 ± 4.04a  93.08 ± 3.71a  WHY -7  100.00 ± 0.00a  97.07 ± 3.19a  93.32 ± 3.02a  Mean values ± standard deviation. a-dMeans within the same column lacking a common superscript differ (P < 0.05). 1The growth rate at 0% bile salts had been used as a control, measured with 100%. View Large Tolerance to Nutrition Starvation The tolerance of the yeast strains to nutrition starvation is given in Table 7. With the prolongation of starvation time, the CFU of all the strains decreased, with WHY-2 and WHY-7 exhibiting the highest number of CFUs on the 10th day of starvation. Table 7. Tolerance of the S. cerevisiae strains to nutrition starvation (107 CFU/mL).   Time/d  Strains  2  4  6  8  10  WHY -1  9.77 ± 0.04b  4.23 ± 0.03c  3.43 ± 0.03c  3.13 ± 0.02b  2.33 ± 0.01b  WHY -2  11.32 ± 0.03a  10.00 ± 0.01a  9.31 ± 0.05a  6.21 ± 0.04a  4.45 ± 0.02a  WHY -3  9.67 ± 0.05b  8.33 ± 0.14b  7.16 ± 0.04b  5.75 ± 0.03a  2.01 ± 0.01b  WHY -4  9.23 ± 0.06b  5.30 ± 0.05c  4.63 ± 0.03c  2.03 ± 0.02c  1.15 ± 0.02c  WHY -5  9.30 ± 0.10b  8.42 ± 0.06b  7.76 ± 0.05b  3.67 ± 0.04b  1.68 ± 0.04a,b  WHY -6  9.97 ± 0.05b  4.43 ± 0.02c  3.67 ± 0.02c  2.93 ± 0.05b  1.03 ± 0.03c  WHY -7  9.63 ± 0.06b  8.30 ± 0.01b  7.96 ± 0.12b  6.16 ± 0.07a  3.63 ± 0.02a    Time/d  Strains  2  4  6  8  10  WHY -1  9.77 ± 0.04b  4.23 ± 0.03c  3.43 ± 0.03c  3.13 ± 0.02b  2.33 ± 0.01b  WHY -2  11.32 ± 0.03a  10.00 ± 0.01a  9.31 ± 0.05a  6.21 ± 0.04a  4.45 ± 0.02a  WHY -3  9.67 ± 0.05b  8.33 ± 0.14b  7.16 ± 0.04b  5.75 ± 0.03a  2.01 ± 0.01b  WHY -4  9.23 ± 0.06b  5.30 ± 0.05c  4.63 ± 0.03c  2.03 ± 0.02c  1.15 ± 0.02c  WHY -5  9.30 ± 0.10b  8.42 ± 0.06b  7.76 ± 0.05b  3.67 ± 0.04b  1.68 ± 0.04a,b  WHY -6  9.97 ± 0.05b  4.43 ± 0.02c  3.67 ± 0.02c  2.93 ± 0.05b  1.03 ± 0.03c  WHY -7  9.63 ± 0.06b  8.30 ± 0.01b  7.96 ± 0.12b  6.16 ± 0.07a  3.63 ± 0.02a  Mean values ± standard deviation. a-cMeans within the same column lacking a common superscript differ (P < 0.05). View Large Table 7. Tolerance of the S. cerevisiae strains to nutrition starvation (107 CFU/mL).   Time/d  Strains  2  4  6  8  10  WHY -1  9.77 ± 0.04b  4.23 ± 0.03c  3.43 ± 0.03c  3.13 ± 0.02b  2.33 ± 0.01b  WHY -2  11.32 ± 0.03a  10.00 ± 0.01a  9.31 ± 0.05a  6.21 ± 0.04a  4.45 ± 0.02a  WHY -3  9.67 ± 0.05b  8.33 ± 0.14b  7.16 ± 0.04b  5.75 ± 0.03a  2.01 ± 0.01b  WHY -4  9.23 ± 0.06b  5.30 ± 0.05c  4.63 ± 0.03c  2.03 ± 0.02c  1.15 ± 0.02c  WHY -5  9.30 ± 0.10b  8.42 ± 0.06b  7.76 ± 0.05b  3.67 ± 0.04b  1.68 ± 0.04a,b  WHY -6  9.97 ± 0.05b  4.43 ± 0.02c  3.67 ± 0.02c  2.93 ± 0.05b  1.03 ± 0.03c  WHY -7  9.63 ± 0.06b  8.30 ± 0.01b  7.96 ± 0.12b  6.16 ± 0.07a  3.63 ± 0.02a    Time/d  Strains  2  4  6  8  10  WHY -1  9.77 ± 0.04b  4.23 ± 0.03c  3.43 ± 0.03c  3.13 ± 0.02b  2.33 ± 0.01b  WHY -2  11.32 ± 0.03a  10.00 ± 0.01a  9.31 ± 0.05a  6.21 ± 0.04a  4.45 ± 0.02a  WHY -3  9.67 ± 0.05b  8.33 ± 0.14b  7.16 ± 0.04b  5.75 ± 0.03a  2.01 ± 0.01b  WHY -4  9.23 ± 0.06b  5.30 ± 0.05c  4.63 ± 0.03c  2.03 ± 0.02c  1.15 ± 0.02c  WHY -5  9.30 ± 0.10b  8.42 ± 0.06b  7.76 ± 0.05b  3.67 ± 0.04b  1.68 ± 0.04a,b  WHY -6  9.97 ± 0.05b  4.43 ± 0.02c  3.67 ± 0.02c  2.93 ± 0.05b  1.03 ± 0.03c  WHY -7  9.63 ± 0.06b  8.30 ± 0.01b  7.96 ± 0.12b  6.16 ± 0.07a  3.63 ± 0.02a  Mean values ± standard deviation. a-cMeans within the same column lacking a common superscript differ (P < 0.05). View Large Determination of Cell Surface Hydrophobicity The results of cell surface hydrophobicity of the yeast strains are shown in Figure 1. Maximum cell surface hydrophobicity of 73.71% was exhibited by WHY-6, followed by WHY-7 and WHY-2. Figure 1. View largeDownload slide Cell surface hydrophobicity of the S. cerevisiae strains. Error bars indicate standard deviations. Figure 1. View largeDownload slide Cell surface hydrophobicity of the S. cerevisiae strains. Error bars indicate standard deviations. Autoaggregation It can be observed from Figure 2 that the autoaggregation percentage for the seven yeast strains ranged from 96.68% (WHY-5) to 98.99% (WHY-2) at 24 h of incubation. At 2 h of incubation, the autoaggregation percentage for the seven strains presented only slight variation, from 24.06% (WHY-1) to 38.86% (WHY-7). In contrast, at 4 h of incubation, all the yeast strains rapidly and automatically aggregated, and the autoaggregation percentage for WHY-2, WHY-5, and WHY-7 was significantly higher, when compared with that for the other four strains. Figure 2. View largeDownload slide Autoaggregation percentage for the S. cerevisiae strains after 2 h (light gray), 4 h (gray), and 24 h (dark gray) of incubation. Error bars indicate standard deviations. Figure 2. View largeDownload slide Autoaggregation percentage for the S. cerevisiae strains after 2 h (light gray), 4 h (gray), and 24 h (dark gray) of incubation. Error bars indicate standard deviations. DISCUSSION Yeasts are one of the important feed probiotics, which are resistant to changes in intestinal pH and can regulate the micro-ecological balance of the intestinal environment and improve animal immune capacity (Garcia-Hernandez et al., 2012; Tomicic et al., 2016). It is generally believed that the best source of an ideal probiotic strain is the intestine of the native animal (Garcia-Hernandez et al., 2012; Zhang et al., 2013; Forkus et al., 2017; He et al., 2017). Yeasts do not belong to the gut-inherent microbial flora, and occur only sporadically in the gut, playing roles in the digestive tract of animals for survival (Binetti et al., 2013; Tomicic et al., 2016). In the present study, 35 yeast strains were isolated from the digestive tract of Cherry Valley ducks collected from four different regions, which suggested that yeasts can tolerate the intestinal environment of meat ducks, but occur as foreign microflora in the intestine. In China, products containing Saccharomyces spp. have been listed as feed ingredients in Feed Ingredients Catalog (2013), indicating that Saccharomyces spp. used in feed are safe. However, the safe strains that can be used in probiotics must undergo a series of screening. As in vivo studies investigating the health benefits of potential probiotics are time-consuming and often expensive, the consequent use of in vitro tests as selection criteria is unavoidable to reduce the number of strains and determine the most effective microorganism (Valeriano et al., 2014; Iaconelli et al., 2015; Forkus et al., 2017). Resistance to pH and bile salts is of significant importance in the survival and growth of microorganisms in the intestinal tract and a prerequisite for probiotics, and S. cerevisiae is well-tolerant to acidic environment (Garcia-Hernandez et al., 2012; Binetti et al., 2013). In the present study, most of the yeast strains were found to survive at pH 3.0–4.0; however, only WHY-2 and WHY-7 presented higher survival rate at pH 2.0. Furthermore, strains WHY-2, WHY-6, and WHY-7 exhibited higher survival rate in the presence of 0.60% bile salts. As animal body temperature is generally about 37°C, limiting temperature is 42°C, and optimal temperature for yeasts is usually 30°C, heat tolerance screening of candidate yeast strains is necessary. In the present study, the strains WHY-2, WHY-6, and WHY-7 survived well at 37°C, while strains WHY-2 and WHY-7 could even survive at 42°C. Furthermore, as the hind gut of animals is often a nutrient-deficit environment, nutrition starvation screening of yeast strains is also vital. The results of the present study showed that the cell counts of all the yeast strains were reduced owing to starvation. However, the strains WHY-2 and WHY-7 exhibited relatively higher cell counts on the 10th day of starvation. Cell surface hydrophobicity is one of the important properties of probiotics, and has been primarily studied based on microbial adhesion to hydrocarbons (Gil-Rodríguez et al., 2015). In the present study, the selected isolates presented strong hydrophobicity in n-hexadecane, indicating potential putative probiotic property (Ogunremi et al., 2015). In particular, the strains WHY-6, WHY-2, and WHY-7 showed higher cell surface hydrophobicity. Another characteristic of a potential probiotic microorganism is the ability to form cellular aggregates, because aggregates can increase microbial adherence to the intestine, thus providing advantages in colonization of the GI tract (García-Cayuela et al., 2014). As yeast cells are bigger and heavier than bacteria, they precipitate quickly and in higher proportion (Gil-Rodríguez et al., 2015). In the present study, the autoaggregation percentages for all the strains were not high at 2 h, but remarkably increased at 4 h of incubation. These results are similar to those reported by Gil-Rodríguez et al. (2015), who demonstrated that the yeasts displayed rapid autoaggregation within the first 4 h of incubation. CONCLUSION In the present study, 35 yeast strains were isolated and identified from the GI tract of ducks, of which seven strains belonged to S. cerevisiae. These seven yeast strains were screened for their cell surface hydrophobicity, autoaggregation ability, and tolerance to high temperature, low pH, bile salts, and nutrition starvation. The results obtained showed that strains WHY-2 and WHY-7 had higher performance, and could be potential probiotic candidates. Thus, the findings of this study could help in selecting probiotic yeast candidates for use in poultry industry. Acknowledgements This work was funded by the Wuhan Science and Technology Bureau, Wuhan city, China (Project 2014020101010073). We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript. Notes The appropriate scientific section for the paper is “Microbiology and Food Safety”. REFERENCES Binetti A., Carrasco M., Reinheimer J., Suarez V.. 2013. 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Poultry ScienceOxford University Press

Published: May 14, 2018

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